In scanning microscopy, a specimen is illuminated with a light beam in order to observe the reflected or fluorescent light emitted by the specimen. The focus of the illuminating light beam is moved in a specimen plane by means of a controllable beam deflection device, generally by tilting two mirrors; the deflection axes are usually perpendicular to one another, so that one mirror deflects in the X direction and the other in the Y direction. Tilting of the mirrors is brought about, for example, by means of galvanometer positioning elements. The power level of the detected light coming from the specimen is measured as a function of the position of the scanning beam. The positioning elements are usually equipped with sensors to ascertain the present mirror position.
In confocal scanning microscopy specifically, a specimen is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto an aperture (called the “excitation pinhole”), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection stop, and the detectors for detecting the detected or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen travels through the beam deflection device back to the beam splitter, passes through it, and is then focused onto the detection pinhole behind which the detectors are located. Detected light that does not derive directly from the focus region takes a different light path and does not pass through the detection pinhole, so that a point datum is obtained which results, by sequential scanning of the specimen, in a three-dimensional image. A three-dimensional image is usually achieved by acquiring image data in layers.
Ideally, the track of the scanning light beam on or in the specimen describes a meander (scanning one line in the X direction at a constant Y position, then stopping the X scan and slewing by Y displacement to the next line to be scanned, then scanning that line in the negative X direction at constant Y position, etc.).
The power level of the light coming from the specimen is measured at fixed time intervals during the scanning operation, and thus sampled one grid point at a time. The measured value must be unequivocally allocated to the scan position associated with it so that an image can be generated from the measured data. It is useful if the status data of the displacement elements of the beam deflection device are also continuously measured for this purpose or, although this is less accurate, if the reference control data of the beam deflection device are used.
Accurate allocation of the position signals to the detected signals is particularly important. In making the allocation, transit time differences and the differing processing times of the detectors acquiring the signals must be taken into account, for example using delay elements. Very stringent stability requirements must be imposed. For example, for an image width of 1000 image points, the transit time fluctuations must remain well below 0.1%.
As the scanning speed becomes greater, the scanning track deviates more and more from the meander shape. This phenomenon is essentially attributable to the inertia of the moving elements. With rapid scanning, the scanning track is more similar to a sine curve, and it often happens that the trajectory portion when scanning in the positive X direction differs from the trajectory portion when scanning in the negative X direction.
German Patent Application DE 197 02 752 discloses a triggering system for a scanner, in particular for a laser scanning microscope, having: an oscillating motor for driving an oscillating mirror that serves to deflect a beam in linearly oscillating fashion; a triggering unit for supplying to the oscillating motor an excitation current that is modifiable in terms of triggering frequency, frequency curve, and amplitude; a function generator that is connected to the triggering unit; and a measured value transducer for obtaining a sequence of data about the deflection positions of the oscillating mirror. The triggering system is characterized in that the measured value transducer is linked to the function generator via a logic unit for ascertaining correction values for the excitation current. It is thereby possible, by evaluating the data made available by the measured value transducer concerning the actual deflection position of the oscillating mirror, to ascertain correction values using the logic unit. Those values can in turn be used to influence the triggering frequencies outputted by the function generator in such a way that the deviations are minimized or entirely eliminated.
The position signal of a positioning element, in particular the position sensor of a galvanometer (actual signal), is affected by interference signals. These interference signals overlie the usable signal and falsify it. If that position signal is then used in a scanning system to determine the position of a scanned data value, it thus results in a position error.
The interference can be divided into several categories: on the one hand there is stochastic interference such as e.g. noise, etc.; on the other hand there is non-stochastic interference such as e.g. incoupling from other sources, distortion, nonlinear transmission effects, etc.
If, in scanning microscopy, an image sequence is generated using the measured values, that sequence then exhibits defects because of the interference. The stochastic interference causes a positional uncertainty, i.e. the position of an image point fluctuates from one image to the next (jitter). The non-stochastic interference can cause positional falsification, which can fluctuate (incouplings) or can be constant over time (distortion, nonlinear transmission effects). The interference on the one hand causes the image sequence to look unsteady, and on the other hand can make it appear distorted. If image data from the forward and return directions of the scan are used, it may happen that the images of structures that occur in two successive lines can no longer be superimposed. Such structures perpendicular to the scanning direction therefore appear to be “fringed.” If the higher-frequency components of the interference spectrum are considered, these may derive only from the measurement system due to the mechanical inertia of the galvanometer.
Thermal and other drift effects cause the delay time between the exciting signal and the position of the galvanometer mirror to change. As a result, with a bidirectional scan the forward and return lines drift apart from one another. This effect must be compensated for manually, in unchangeable fashion. This effect occurs in particular when a generated position signal is used to determine the position of the scan data.